Autophagy assay cell type - U87MG

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 8 matching solutions for this experiment

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.

Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.
Upstream tips
Intercalates between DNA/RNA and used to analyze mitochondria and Lysosomal content by flow cytometry.
Protocol tips
Stain cells with acridine orange (1 μg/ml) for 15 mins at 37°C
Upstream tips
Highly potent inhibitor for mTOR and DNA-PK while inhibits PI3-K only at much higher concentration
Protocol tips
Culture the cells in DMEM medium containing 250nM Torin1
Upstream tips
FDA approved Cox-2 inhibitor. It is used to induce autophagy and apoptosis in cells.
Protocol tips
Treat cells with 30 μM of celecoxib for 72 hours
Upstream tips
It is an autophagosome inhibitor.
Protocol tips
Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line
Upstream tips
It is an autophagosome inhibitor.
Protocol tips
Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line
Dansylcadaverine

Sigma-Aldrich

Protocol tips
Incubate with 50 μM of MDC in PBS 1x at 37 °C for 15 min
Anti-LC3 pAb

MBL international corporation

Protocol tips
For Co-IP, Use non-denaturating lysis solution to lyse the cells and incubate the cell lysate for 3 hrs at 4°C with Anti LC3 Antibody.Protein A/G then can be incubated overnight.
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!
Become shareholder Discussions About us Contact Privacy Terms